Abstract The long-term objective of the proposed research is to elucidate the mechanisms of xenobiotic- mediated inactivation, degradation, and turnover of cytochrome P450 enzymes. Nitric oxide synthase (NOS), the most highly regulated cytochrome P450 enzyme, plays a key role in a variety of biological processes, including regulation of gastrointestinal motility and liver drug metabolism. We have discovered that drugs, such as guanabenz and tobacco, are metabolism- based inactivators of neuronal NOS (nNOS) and lead to the covalent alteration, enhanced turnover, and loss of nNOS P450 protein via the ubiquitin proteasomal pathway. The loss of NOS is a mechanism of toxicity associated with these drugs. We have established that alteration of the active site conformation `labilizes' the nNOS, which is then recognized by Hsp70 and Hsp90 chaperones, and is ubiquitinated by CHIP, a chaperone-associated ubiquitin ligase, resulting in the specific proteasomal degradation of the labilized nNOS. We plan on utilizing these discoveries and our recent ground-breaking success with electron microscopy (EM) studies on nNOS and nNOS?Hsp70?CHIP complexes to better understand how chaperones recognize labilized nNOS P450 through the following specific aims: (1) To characterize the structures of the stabilized and labilized states of nNOS with the use of single particle negative stain EM and cryogenic-EM techniques, (2) To characterize the structure of nNOS chaperone complexes with Hsp70 and Hsp90 by EM as well as LC-MS/MS techniques, (3) To isolate and characterize the chaperones, co-chaperones and other proteins that associate with labilized nNOS by use of a cell permeable thiol-cleavable crosslinker and LC- MS/MS methods. This work would be the first to elucidate the structure of full-length nNOS, nNOS?chaperone complexes, as well as determine the specific conformational states of nNOS that are recognized by chaperones. These studies should lead to a better understanding of how chaperones recognize labilized forms of nNOS and maintain protein quality. Ultimately, these studies may provide a way to predict, evaluate, and refine, the efficacy and safety of drugs and other xenobiotics. Moreover, understanding the mechanism of recognition of labilized nNOS and quality control may provide a new method to specifically remove proteins for therapeutic benefit. An example of such utility is our recent study on removal of protein aggregates through activation of chaperones in a neurodegenerative disease model (Nature Chemical Biology 9: 112-118, 2013).